Catalytic mechanism of the zinc-dependent MutL endonuclease reaction

The catalytic mechanism for the DNA cleavage activity of the mismatch repair system endonuclease MutL has been identified, which provides a clue as to how cadmium inhibits its activity.


Introduction
DNA mismatch repair (MMR) system corrects DNA mismatches generated by errors in DNA replication, recombination among nonidentical sequences, and other processes (1,2,3,4,5). Because MMR significantly contributes to maintain genome integrity, mutations in bacterial MMR genes are related to an increase in antibiotic resistance (6), and mutations and epigenetic silencing of human MMR genes can be causative of a hereditary cancer, Lynch syndrome (7,8,9).
In the MMR pathway, bacterial MutS and its eukaryotic counterparts (MutSα and MutSβ) recognize DNA mismatches and subsequently recruit bacterial MutL and eukaryotic counterparts such as MutLα, respectively (3,10,11,12,13). MutLs, with a few exceptions, are endonucleases that incise the error-containing (i.e., newly synthesized) strand of the mismatched duplex (14,15,16,17). Prokaryotic MutL endonucleases are homodimers, whereas eukaryotic MutL endonucleases are heterodimers, with only one of the two subunits having endonuclease activity. The bacterial DNA polymerase III β subunit (β-clamp) and its eukaryotic counterpart PCNA interact with MutL, which directs the incision to the newly synthesized strand (18,19,20,21). MutL-dependent incision induces downstream MMR reactions, where single-stranded DNA-specific exonucleases excise the error-containing strand to generate a gap region, replicative DNA polymerases fill the gap, and DNA ligases seal the nick to complete the reaction.
Prokaryotic MutL endonucleases and the endonuclease subunits of eukaryotic MutL homologs include two domains: the N-terminal ATPase domain and the C-terminal endonuclease domain (CTD) (22,23). Crystallographic analyses have revealed that CTD consists of the regulatory and dimerization subdomains. The regulatory subdomain is missing in MutLs from some bacterial species including Aquifex aeolicus, whereas the dimerization subdomain is found in all MutL endonucleases (24). The dimerization subdomain contains the metal-binding sites, in which multiple zinc ions are coordinated by the residues from highly conserved motifs: DQHAX 2 EX 4 E, ACR, and CPHGRP (underlined residues coordinate the zinc ions) (23,25,26,27,28). These motifs are conserved in all MutL endonucleases including eukaryotic MutLγ, another MutL homolog that participates in both MMR and crossover formation during meiosis (29,30,31,32), but not conserved in MutLs without endonuclease activity (33,34).
Human and yeast MutLα endonucleases exhibit manganesedependent activation (14,15). Similar finding was observed with MutLs from Bacillus subtilis, Thermus thermophilus, A. aeolicus, and other bacteria (23,35,36). In the crystal structure of the B. subtilis MutL CTD, zinc ions were observed only when the crystallization condition contained zinc ions (23). One of the two binding sites had a lower affinity to zinc ion and was presumed to have a structural function rather than a catalytic one. This was later supported by structural analysis of the A. aeolicus MutL (aqMutL) CTD in its single or multiple zinc-bound states, where multiple zinc ions maintained the conformation of the CTD (37). It has been confirmed that the Asp residue at the beginning of the DQHAX 2 EX 4 E motif is critical for the nuclease activity of eukaryotic and bacterial MutLs (14,23,35), although the Asp residue is not involved in zinc binding. Therefore, it is inferred that another metal ion, such as a manganese ion, acts as a catalytic metal ion and is coordinated by the Asp residue (38).
On the other hand, Gueneau et al have proposed another model based on the crystal structure analyses of the yeast MutLα CTD (38). The yeast MutLα CTD bound zinc ions at two binding sites even without adding zinc ions during the protein preparation and crystallization procedures (25). The arrangement of the two zinc ions is reminiscent of the two-metal-ion mechanism that is widely adopted in phosphoryl transfer enzymes. The same arrangement was also observed for the yeast MutLγ CTD (39). These imply that the two zinc ions are the catalytic metal ions for MutL endonucleases. This hypothesis is supported by the recent finding that human MutLα exhibited the nuclease activity with zinc ions alone (40). However, the result does not rule out the possibility that a catalytic zinc ion binds to an unknown site such as the Asp residue of the DQHAX 2 EX 4 E motif.
Cadmium exhibits chemical similarities to zinc and is known to replace zinc in many metalloenzymes (41). Incorporation and accumulation of cadmium within human bodies is associated with several types of cancer; therefore, cadmium is classified as a carcinogen for human (42). In addition, the mutagenic effect of cadmium on mammalian cells has also been reported (43). The mutagenic effect of cadmium has been linked to specific inhibition of the MMR activity (44). Recently, we found that cadmium ions bind to the same sites of the aqMutL CTD as zinc ions (27, 37). Furthermore, Sherrer et al revealed that cadmium ion specifically interfered with the endonuclease activity of human MutLα (40). However, the detailed mechanism of inhibition by cadmium remains unclear as the catalytic mechanism of the nuclease activity of MutL is not yet understood.
In this study, we first solved the crystal structure of the aqMutL CTD in the manganese ion-bound state. Manganese ions were located at the same binding sites as zinc ions, which indicates that the nuclease catalytic site is defined by these zinc-binding sites. Mass spectrometric analysis revealed that aqMutL yielded the 59phosphate and 39-OH products, which is characteristic of the twometal-ion mechanism. By comparing the zinc-and cadmium-bound structures of the aqMutL CTD, we found that the position and flexibility of the Arg residue of the CPHGRP motif were affected by binding cadmium ions. Site-directed mutagenesis and an in vivo complementation assay indicated that the Arg residue plays a catalytic role in the nuclease reaction. From these and other results, we propose that MutL incises DNA by the two-metal-ion mechanism, where the positive charge of the Arg residue of the CPHGRP motif is catalytically important. The mutagenicity of cadmium might be explained by the cadmium-dependent miscoordination of the catalytic Arg residue.

Results and Discussion
Manganese ions bind to the same sites of aqMutL as zinc ions Previously, we determined the crystal structure of the zinc ionbound form of the aqMutL CTD, where three zinc ions were identified at the M1, M2, and M3 sites (Fig 1A and B) (37). The M1 and M2 sites are defined by the DQHAX 2 EX 4 E, ACR, and CPHGRP motifs and are identical to the zinc ion-binding sites of the yeast MutLα and B. subtilis MutL CTDs (23,25). The M3 site is unique to aqMutL and required to maintain the open conformation of the active site (37).
The observations of the manganese ion-dependent activation, the findings supporting structural functions for zinc ion, and the presence of a critical Asp residue outside the M1-M3 sites imply the possibility that MutL has another site accommodating a catalytic metal ion. In order to explore this possibility, we performed crystal structure analysis of the aqMutL CTD complexed with manganese ions. The aqMutL CTD, which is purified as the zinc-bound form, was incubated with the buffer solution containing 2 mM manganese ion at 70°C for 24 h. After dialysis against the same buffer at 25°C for 24 h, the absence of zinc was confirmed by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The protein was concentrated and crystallized in the presence of a 50 mM manganese ion. The presence of manganese ions and the absence of zinc ions in the crystal were confirmed by the manganese K-edge and the zinc K-edge X-ray absorption fine structure analyses, respectively ( Fig S1A and B). The crystal structure was solved with manganese single-wavelength anomalous dispersion phasing (Table S1). Peaks of electron density in the anomalous diffraction map calculated from data collected at a wavelength of 1.8900Å were clearly observed at the M1, M2, and M3 sites of the aqMutL CTD at 100% occupancy ( Fig 1C). The wavelength of 1.8900Å was chosen to be above the absorption edge of manganese so that the observed peaks of anomalous diffraction correspond to manganese atoms. The overall structure of the manganese-bound form was almost identical to that of the zinc-bound form; the root-meansquare deviation between the two structures was 0.1Å ( Fig 1D).
No peak of electron density in the anomalous diffraction map was observed around Asp351 of the DQHAX 2 EX 4 E motif. Additional peaks were found at the other sites M4, M5, and M6 that were coordinated by Asp366, Glu384, and Glu416, respectively ( Fig S2A). These acidic residues are not conserved in other MutL homologs and, in addition, were confirmed to not be required for the manganese-dependent endonuclease activity of aqMutL ( Fig S2B). Therefore, metal ions at the M4, M5, and M6 sites are not the catalytic metal ions, which would be artifacts observed because of high concentrations of manganese ions in the preincubation and crystallization conditions. Thus, it was confirmed that manganese ions bind to the same sites of aqMutL as zinc ions. Together with experimental results from previous studies identifying catalytically important residues (14,15,34), the M1 and M2 sites are likely to be the binding sites for the catalytic metals.
As Gueneau et al have pointed out, arrangements of the M1 and M2 zinc ions of MutL are similar to those of nucleases that employ the two-metal-ion mechanism (25,38). To examine the possibility that the two-metal-ion mechanism works in the MutL endonuclease reaction, properties of the products of the aqMutL nuclease reaction were characterized. The 16-bp double-stranded DNA was degraded by aqMutL (Fig 2A), and the reaction products were purified and analyzed by MALDI TOF/TOF mass spectrometry (MS). As shown in Fig 2B, a series of degradation fragments was detected. The cleavage products were completely dissociated into single-   stranded DNAs during the MS analysis. The masses of the detected fragments were consistent with the theoretical masses with errors of less than 100 ppm ( Table 1), assuming that aqMutL leaves 59phosphate and 39-OH groups at the cleavage site. Although fragments with a 59-phosphate group could not be efficiently fragmented by our MS/MS analysis, those with a 39-OH group were precisely identified by the MS/MS analysis (Fig S3A-C). Thus, it was revealed that aqMutL digested the DNA at the 39-side of the phosphodiester bond. This is characteristic of cleavage by the two-metal-ion mechanism (45).

Cadmium ion limits the flexibility of the side chain of Arg406
In the reaction by the two-metal-ion mechanism, two metal ions act as a general base and a Lewis acid to activate the attacking water molecule and stabilize the negatively charged transition state, respectively (46,47). In addition to amino acid residues involved in the coordination of these catalytic metal ions, other amino acid residues are often involved in the catalysis by assisting the functions of the metal ions. In order to explore the possible catalytic residues of MutL, we focused on the inhibitory effect of cadmium on MutL endonuclease activity. It was recently found that cadmium was a specific inhibitor of the endonuclease activity of human MutLα by competing with zinc ions (40), which would be one of the mechanisms of the carcinogenic effect of cadmium.
To test whether cadmium interferes with the endonuclease activity of aqMutL, zinc ions in the purified aqMutL sample were replaced with cadmium ions. The protein solution was incubated in the buffer containing 0.5 mM zinc or cadmium ions at 60°C for various periods of time, and the endonuclease activity of aqMutL was measured. As shown in Fig 3A and B, the activity was significantly reduced after 12 h incubation with cadmium ion, indicating that cadmium ions gradually replaced zinc ions and interfered with the endonuclease activity of aqMutL. The DNA binding of aqMutL and zinc-bound (magenta) forms of the aqMutL CTD. The cadmium-bound and zinc-bound forms were superimposed so that the positions of the three metals and the metal-coordinating residues of the subunit on the right side coincide between the two forms. Metal ions are shown in sphere models. Gly422 is located at the tip of the loop structure extending from the α4 helix of the other subunit. The α4 helix in the cadmium-bound form moved by 2.70Å compared with that in the zinc-bound form.
(H) Superimposition of the dimeric structures of the cadmium-bound (gold/orange) and zinc-bound (light pink/magenta) forms of the aqMutL CTD. (C, E, F) It should be noted that, of the two catalytic sites shown here, the one on the right side (indicated by a red arrow head) is shown in panels (C, E, F). (I) A schematic representation of cadmium-dependent structural changes in the aqMutL CTD, which depicts the movement of the α4 helix (black arrow) and the change in the subunit arrangement (red arrow). Source data are available for this figure.
How cadmium interferes with MutL Fukui et al.
https://doi.org/10.26508/lsa.202302001 vol 6 | no 10 | e202302001 was not altered by the presence of cadmium ions, suggesting that the catalytic step of the endonucleolytic reaction was specifically disrupted by the ions (Fig S4A and B). We previously found that cadmium ions bound to the same sites as zinc ions in the aqMutL CTD (27, 37). The gross structure of the cadmium-bound form of the aqMutL CTD was almost identical to that of the zinc-bound form. However, reanalysis of the detailed structures revealed that there were some differences between the two forms: High-resolution structures enabled us to find that the electron density for the guanidino group of Arg406 was missing in the zinc-bound form, whereas that was clearly seen in the cadmium-bound form (Fig 3C and  D). The side chain of Arg406 was fixed by the M1 cadmium ion via a water molecule, W1. The distance between the M1 cadmium ion or zinc ion and the W1 water molecule was 2.53 or 2.24Å, respectively (Fig 3E and F). In the cadmium-bound form, the side chain of Arg406 also interacted with the main-chain carbonyl group of Gly422 from the other subunit of the dimer (Fig 3E), causing a movement of the α4 helix (Figs 3G and  S5). The movement of the α4 helix accompanied a change in the subunit arrangement of the aqMutL CTD dimer (Fig 3H and I). As the result, in the cadmium-bound form, Gly422 (carbonyl oxygen atom) was~1.0Å closer to Arg406 (Cδ atom) than in the zinc-bound form (Fig S6A). Despite these structural changes, no significant difference was found in the structure around the metal-binding site except for the side chain of Arg406 (Fig S7).
To confirm that the observed differences depend on the metal species, cadmium ions in the crystal were replaced with zinc ions. The crystal of the cadmium-bound form was washed with and soaked in the crystallization buffer containing 50 mM Zn 2+ for 2 d. The same process was repeated three times. Replacement of cadmium ions with zinc ions was confirmed by the zinc K-edge X-ray absorption fine structure analysis (Fig S1C). The zinc-restored structure of the aqMutL CTD was solved by Zn-SAD phasing (Table S1). The result demonstrated that the electron density for the guanidino group of Arg406 was missing in the zincrestored form (Fig 4A, left panel) and that the distance between the M1 zinc ion and the W1 water molecule was 2.24Å ( Fig 4A, right  panel). The position of the α4 helix in the zinc-restored form was identical to that in the zinc-bound (zinc-cocrystallized) form ( Fig 4B). Furthermore, the structure of Arg406 and the quaternary structure of the zinc-restored form were also identical to those of the zinc-bound form (Figs S6B and S8, respectively). This is in good agreement with the previous report that inhibition of human MutLα by cadmium ion could be rescued by addition of zinc ion (40).
The electron density of the guanidino group of Arg406 was also missing in the manganese-bound form of the aqMutL CTD (Fig 4C,  left panel and Fig S6E). The distance between the M1 manganese ion and the W1 water molecule was 2.25Å (Fig 4C, right panel). Likewise, the position of the α4 helix, the structure of Arg406, and the quaternary structure of the manganese-bound form were the same as those in the zinc-bound form (Figs 4D, S6C, and S8, respectively). These are consistent with the fact that manganese is catalytically competent in the endonuclease activity of MutL. However, because no structural differences were observed between the manganesebound and zinc-bound forms, it could not be explained why manganese activates the activity more strongly than zinc.  The R847T mutation has been found in Lynch syndrome-suspicious patients and registered as a variant of uncertain significance at the ClinVar database (48). In order to test the importance of Arg406 in the endonuclease activity, the R406A, R406T, and R406K mutant forms of the aqMutL were prepared. The circular dichroism spectra of the mutant forms were identical to that of the WT aqMutL (Fig S9), indicating that the mutations did not affect the integrity of the protein structure. The DNA-binding ability of aqMutL was not influenced by these mutations (Fig 5B and C), whereas the endonuclease activity was severely impaired by the R406A and R406T mutations (Fig 5D and E). The R406K mutant form retained significant, albeit weaker, endonuclease activity. These results suggest that the Arg residue in the CPHGRP motif is a catalytic residue for the endonuclease activity. The R406T mutant form of the aqMutL CTD was crystallized in the presence of cadmium ion, and the structure was solved with the Cd-SAD phasing (Table S1). As was expected from the CD measurements, the overall structure was almost identical to that of the WT aqMutL CTD. The distance between the M1 cadmium ion and the W1 water molecule was 2.57Å ( Fig 5F). However, the position of the α4 helix, the local structure around Thr406, and the quaternary structure were the same as those of the zinc-bound form of the WT aqMutL CTD (Figs 5G, S6D, and S8, respectively). These results support the notion that the M1 cadmium ion fixes the side chain of Arg406 of aqMutL, causing the shift in the position of the α4 helix and the change in the quaternary structure.
The importance of Arg406 for the repair activity in the cell was examined by phenotype complementation experiments using a eubacterium, T. thermophilus, one of the model organisms that have been used for studies on DNA repair including MMR (49). Disfunction of MMR causes a significant increase in mutation rate that can be determined by measuring the frequency of the streptomycin-resistant bacterial cells. The CPHGRP and other metal-binding motifs are also conserved in T. thermophilus MutL, with Arg500 corresponding to Arg406 of aqMutL. As shown in Fig 5H, introduction of the gene for the R500T T. thermophilus MutL did not complement the hypermutator phenotype of the mutL-lacking strain, indicating that the Arg residue in the CPHGRP motif is essential for in vivo MMR. This is consistent with a previous report that the replacement of the corresponding Arg residue with Glu abolished the in vitro MMR activity in the human cell extract (34). Thus, the result of the in vivo complementation experiment reinforces the interpretation that this Arg residue plays a catalytic role in the endonuclease activity of MutL. The gene encoding the R500K T. thermophilus MutL did not complement the hypermutator phenotype (Fig 5H), although the corresponding mutant form, the R406K, of aqMutL retained the endonuclease activity. Even a slight decrease in the endonuclease activity of MutL might be sufficient to abolish the in vivo MMR activity of T. thermophilus, which is consistent with the fact that an Arg residue at this position is conserved in MutLs from many biological species.

Catalytic mechanism of the MutL endonuclease activity
The present crystallographic and other experiments suggest that the endonuclease reaction of MutL is catalyzed by the two-metalion mechanism, where metal ions at the M1 and M2 positions play a central role to cleave the phosphodiester bond of DNA backbone. Although the Asp residue of the DQHAX 2 EX 4 E motif has been known to be critical for the endonuclease activity, our crystallographic results revealed that the residue was not involved in the coordination of the catalytic metal ions. As previously suggested by Guarné and Charbonnier (38), Asp351 might be indirectly involved in the reaction by stabilizing the DQHAX 2 EX 4 E motif-containing helix through the N-capping mechanism.
Results of both in vitro and in vivo experiments indicated that Arg406 is a catalytic residue in the endonuclease activity of aqMutL. Because the R406K mutant form of aqMutL retained significant endonuclease activity, it is thought that a positively charged amino acid residue at the position is important for the activity. Arg406 may have a role as a Lewis acid that, along with the catalytic zinc ions, stabilizes the negatively charged transition state generated by the attack of a nucleophilic water molecule. Further structural analyses on the MutL-DNA complex would help address the details of the role of the Arg residue.

A mechanistic insight into the inhibitory effect of cadmium
In the two-metal ion mechanism, water molecules that are coordinated by the metal ions participate in the catalysis. The reactivity of the coordinated water molecules depends on the valence and radius of the coordinating metal ions. For metal ions of the same valence, the smaller the ionic radius, the smaller the pK a value of the coordinated water molecule, hence the stronger the nucleophile. The radius of hexacoordinated Cd 2+ (0.95Å) is larger than that of hexacoordinated Zn 2+ (0.74Å), which might explain the inhibitory effect of cadmium on the endonuclease activity of MutL. However, this idea seems to be inconsistent with the fact that Mn 2+ acts as a much stronger activator than Zn 2+ while having a larger ionic radius (0.83Å) than Zn 2+ .
Alternatively, the inhibitory effect of cadmium might be explained by the cadmium-dependent structural changes in the catalytic site. In the present study, it was confirmed that a cadmium ion fixed the guanidino group of a catalytic Arg residue of aqMutL. Furthermore, the fixed side chain of the residue interacted with a Gly residue, pulling the α4 helix of the other subunit. The positional change of the α4 helix only slightly affected the structure around the metalbinding site because of a concomitant change in the quaternary structure ( Fig S6A). The restricted flexibility of the side chain of the catalytic Arg residue could be responsible for the inhibitory effect of cadmium.
Interestingly, we found that the R406K mutant form of aqMutL was resistant to the inhibitory effect of cadmium (Fig S10). ICP-AES measurements confirmed that zinc ions were completely replaced with cadmium ions in the R406K mutant form, as in the WT aqMutL (Table S2). The activity of the cadmium-bound form of the R406K aqMutL decreased to, but still retained, 60% of that of its zincbound form, indicating that cadmium is not optimal but inherently functional as a catalytic metal ion of aqMutL. This finding further supports the structural mechanism for the inhibitory effect of cadmium. Because the side chain of the Lys residue is shorter than that of the Arg residue and lacks the guanidino group, it is speculated that the binding of cadmium ions neither fixes the Lys residue in the same way as it does the Arg residue nor alters the position of the α4 helix because the Lys residue cannot bridge the cadmium ion and Gly422. If MutL had evolved with a Lys residue at this position as the catalytic residue, MutL might not have been inhibited by cadmium. Although the structure of the cadmiumbound R406K aqMutL CTD should be helpful to confirm these speculations, we could not obtain the crystal of this mutant form of CTD under the conditions examined.

Plasmid constructions and preparation of proteins
The expression plasmids for the R406A, R406T, and R406K forms of fulllength aqMutL were constructed by introducing the mutations into the pET-11a/aqMutL plasmid using PrimeSTAR mutagenesis (Takara). The WT and mutant forms of the full-length aqMutL and the aqMutL CTD were overexpressed in E. coli and purified by the previously described procedures (27). The protein solutions were concentrated and stored at 4°C.

Crystallography of the manganese-bound form of the aqMutL CTD
The purified aqMutL CTD (20 μM) was incubated in the buffer containing 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM DTT, 10% glycerol, and 2 mM MnCl 2 at 70°C for 24 h. After dialysis against the buffer containing 50 mM HEPES-KOH (pH 7.5), 10% glycerol, and 2 mM MnCl 2 at 25°C for 24 h, the protein was concentrated to 28.5 mg/ml. A 1 μl aliquot of the protein solution was mixed with an equal volume of the crystallization buffer containing 200 mM sodium acetate (pH 4.6), 50 mM MnCl 2 , and 30% (vol/vol) polyethylene glycol 400. The drop was equilibrated against 50 μl of the same buffer at 20°C for 2 d. The crystal of the aqMutL CTD was cryocooled at −173°C. The diffraction data were collected at the wavelength 1.8920Å for the manganese single-wavelength anomalous dispersion phasing and 1.0000Å for the refinement of the model structure at SPring-8 BL38B1 (Hyogo). The diffraction data were processed by the HKL2000 program package (50). Identification of the locations of manganese atoms, initial phasing, phase improvements by density modification, and model building were performed using diffraction data of 1.8920Å wavelength by PHENIX program (51). The model was refined using diffraction data of 1.0000Å wavelength by COOT (52) and PHENIX programs. The statistics for data collection and refinement are shown in Table S1.

Crystallography of the zinc-restored form of the aqMutL CTD
The crystal of the cadmium-bound form of the aqMutL CTD was obtained by the previously described Method (27). The cadmium-bound crystal was washed with the buffer containing 200 mM sodium acetate (pH 4.6) and 30% (vol/vol) polyethylene glycol 200 and soaked in the buffer containing 200 mM sodium acetate (pH 4.6), 50 mM ZnCl 2 , and 30% (vol/vol) polyethylene glycol 400 for 2 d at 20°C. The same process was repeated three times. The crystal was cryocooled at −173°C, and the diffraction data were collected at the wavelength 1.2820Å for the Zn-SAD phasing and 1.0000Å for the refinement of the model structure at SPring-8 BL38B1. The data processing and refinement procedures were the same as those for the manganese-bound crystal of the aqMutL CTD.

Crystallography of the cadmium-bound form of the R406T aqMutL CTD
The cadmium-bound form of the R406T aqMutL CTD was created by the same procedure as that for the WT aqMutL CTD (27). The crystal was cryocooled at −173°C, and the diffraction data were collected at the wavelength 1.5000Å for the Cd-SAD phasing and 1.0000Å for the refinement of the model structure at SPring-8 BL38B1. The data processing and refinement procedures were the same as those for the manganese-bound crystal of the aqMutL CTD.

Inhibition of the endonuclease activity of aqMutL by cadmium
The WT or R406K mutant form of aqMutL (50 μM) was incubated in the buffer containing 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM DTT, 10% glycerol, and 0.5 mM ZnCl 2 or CdCl 2 at 60°C for 0, 3, 6, 12, or 24 h. The supercoiled plasmid DNA (pT7Blue) (50 ng/μl) was incubated with 0-5 μM aqMutL of the WT or R406K mutant form in 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM DTT, and 10% glycerol at 70°C for 30 or 15 min. Protein concentrations used in each experiment were indicated in the panel or the legends for figures. The reactions were stopped by adding 5 × loading buffer (10 mM EDTA, 1% SDS, 50% glycerol, and 0.05% bromophenol blue). Reaction solutions were loaded onto a 1.0% agarose gel containing TBE buffer and electrophoresed with the same buffer. DNA fragments were stained with SYBR Gold and detected under ultraviolet light at 365 nm. The amounts of the signals for the intact supercoiled and the incised DNAs were measured by using CS Analyzer 3.0 (ATTO).
Exchange of zinc for cadmium in the experiment was confirmed by ICP-AES (Table S2). Proteins (50 μM) were incubated with ZnCl 2 or CdCl 2 under the same condition as above for 24 h and dialyzed against the buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 10% glycerol. After filtration, protein concentrations were determined on the basis of the values of absorbance at 280 nm, which indicated that precipitation of the protein during the manipulation was negligible. The concentrations of zinc and cadmium in the protein solutions were measured by an ICP-emission spectrometer PS3520UVDD II (Hitachi High-Tec).
Assay of the endonuclease activity of the D366A, E384A, and E417A mutant forms of the aqMutL CTD using the plasmid DNA In order to examine the manganese-dependent endonuclease activity of the aqMutL CTD, 50 ng/μl supercoiled plasmid DNA (pT7Blue) was incubated with 0-3 μM of the WT or the mutant forms of the aqMutL CTD in the buffer containing 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM DTT, 10% glycerol, and 0.5 mM MnCl 2 at 60°C for 10 min. The amounts of the signals for the intact supercoiled and the incised DNAs were measured by the same procedure as above.
Assay of the endonuclease activity of the R406A, R406T, and R406K mutant forms of aqMutL using the linear double-stranded DNA The 59-fluorescein-labeled 60-mer single-stranded DNA (59-AGGAACCTCGAGGGATCCGTCCTAGCAAGCCGCTAGAGGAACATATCCTTAA GAGTTCCA-39) (eurofins Genomics) was hybridized with the nonlabeled complementary strand (59-TGGAACTCTTAAGGATATGTTCC TCTAGCGGCTTGCTAGGACGGATCCCTCGAGGTTCCT-39) to obtain the labeled 60-bp double-stranded DNA. The labeled DNA was incubated with 0-700 nM of the WT or the mutant forms of aqMutL in the buffer containing 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 0.5 mM MnCl 2 , 1.25 mM MgCl 2 , 1 mM DTT, and 10% glycerol at 55°C for 60 min. The reaction was stopped by the addition of an equal volume of the sample buffer containing 5 mM EDTA, 80% deionized formamide, 10 mM NaOH, 0.1% bromophenol blue, and 0.1% xylene cyanol. The reaction mixtures were loaded onto a 15% acrylamide gel containing 8 M urea and TBE buffer and electrophoresed with the same buffer. DNAs were visualized by using LuminoGraph I (ATTO). The intensities of the signals for the intact and incised DNAs were determined by CS Analyzer 3.0.

Electrophoretic mobility shift assay
The fluorescently labeled double-stranded DNA was generated by hybridizing the 59 Cy5-labeled 60-mer single-stranded DNA (59-AGGAACCTCGAGGGATCCGTCCTAGCAAGCCGCTGCTACCGGAAGCTTCTC-GAGGTTCCT-39) (BEX Co.) with the non-labeled 60-mer singlestranded DNA (59-AGGAACCTCGAGAAGCTTCCGGTAGCAGCGGCTTGCTA GGACGGATCCCTCGAGGTTCCT-39) (BEX Co.). The DNA (50 nM) was incubated with 0-0.7 μM aqMutL in the buffer containing 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 2.5 mM MgCl 2 , 0.1 mM DTT, and 10% glycerol at room temperature for 15 min. The mixtures were loaded onto a 10-20% gradient polyacrylamide gel (ATTO) and electrophorezed in EzRun TG buffer (ATTO). DNAs were visualized by using LuminoGraph I. The intensities of the signals were determined by CS Analyzer 3.0. The %Shifted valules were calculated as percentage of shifted signals to all signals in each lane, i.e., %Shifted = 100 × S shifted /(S free + S shifted ), where S shifted and S free are the amounts of shifted and free DNA signals in each lane.

Circular dichroism spectrometry
Circular dichroism measurements were carried out with a Jasco spectropolarimeter, model J-720W (Jasco). Measurements were performed in a solution containing 30 mM Tris-HCl (pH 8.0) and 10 μM protein using a 0.1 cm cell at 25°C. The residue molar ellipticity [θ] was defined as 100θ obs /(lc), where θ obs is the observed ellipticity, l is the length of the light path in centimeters, and c is the residue molar concentration of the protein.
The reactions were stopped by heat treatment at 98°C for 20 min. The digested fragments in the reaction mixtures were trapped by the SuperQ resin. After washing with water, the fragments were eluted with 0.75 M ammonium acetate. Eluted DNAs were dried up in tubes and redissolved in water just before the MS experiment. The DNA fragments were mixed with 50% acetonitrile containing 10 mg/ ml 3-hydroxypicolinic acid and loaded on a sample plate. MS spectra were measured using a RapifleX, MALDI TOF/TOF instrument (Bruker Daltonics Inc.). The m/z values were externally calibrated by peptide II and protein I standards (Bruker Daltonics Inc.).
Complementation experiment with fluctuation assay T. thermophilus HB8 strain was grown at 70°C in TR medium: 0.4% tryptone (Difco), 0.2% yeast extract (Oriental Yeast), and 0.1% NaCl (pH 7.5). The TR medium was supplemented with 0.4 mM CaCl 2 and 0.4 mM MgCl 2 (TT medium) when used for transformation experiments. The mutL-lacking strain of T. thermophilus HB8 was constructed as previously described (35). The WT, R500T, and R500K T. thermophilus mutL genes were ligated into the pMK18::HygΔKan plasmid using XbaI and HindIII restriction sites. The mutL-lacking strain of T. thermophilus HB8 was transformed with these plasmids. The mutation rate of T. thermophilus HB8 cells was estimated by the Luria-Delbruck fluctuation assay as described previously (53,54). Single colonies of T. thermophilus HB8 on TR plates were inoculated into TR media and cultured at 70°C overnight. The overnight culture was diluted 500-fold with TR medium. The diluted culture (100 μl) was inoculated into 4 ml of TR medium and further incubated at 70°C overnight. The overnight culture was diluted 500-fold with TR medium. Aliquots of the diluted culture (1.5 ml) were dispensed into 20 test tubes and incubated at 70°C for 24 h. Three of the 20 cultures were diluted 10 5 -fold. The diluted culture (50 μl) was plated on the non-selective TR plate and incubated at 70°C for 16 h. The colony-forming units were counted to determine the final population size (Nt). The rest of the 20 cultures were plated on the selective TR plate containing 100 μg/ml streptomycin and incubated at 70°C for 30 h. The colony-forming units were counted, and the number of mutation events (m) were calculated by the program rSalvador (55). The mutation rate of each strain was estimated as m/Nt.